The long term objective of this application is to elucidate pathways for generating an MHC molecule/peptide complex within an antigen presenting cell. The specific aims of this proposal are to functionally and genetically characterize a group of mutant antigen presenting cells, which are unable to present a variety of soluble antigens to class II restricted T cells but can present a peptide antigen. The approaches are: to use proteolytic digests of antigen and DR restricted peptide specific T cell clones to more extensively evaluate the mutants' ability to present antigen; to determine whether the mutants are defective in antigen processing via the endocytic pathway; to use somatic cell hybrids to determine how many complementation groups exist among 8 obligate independent presentation defective mutants; to determine whether human non-lymphoid cells and murine lymphoid cells can complement the defect(s) in the mutants; to determine whether the affected gene(s) map to X chromosome or the HLA-D region of chromosome 6; and to isolate phenotypically similar mutants using retroviral insertional mutagenesis as a tool for eventual cloning of the mutant gene(s). In addition, isolation of other antigen processing mutants by immunoselection with antigen specific cytolytic T cells will be pursued. These studies may lead to a better understanding of MHC restricted antigen processing/presentation and may thus provide insights into the pathophysiology of HLA associated diseases in which MHC restricted T cells are likely to play a role.